Francisella tularensis is a highly virulent pathogen for humans and other mammals. Two clinically important subspecies are recognized, F. tularensis subs, tularensis (type A F. tularensis), and subs holarctica (type B F. tularensis). Although both subspecies cause severe infection in humans, only type A strains are routinely lethal if left untreated, especially following inhalation of the pathogen. An empirically attenuated type B strain of the pathogen, F. tularensis live vaccine strain (F. tularensis LVS) has served as a useful vaccine against type A F. tularensis for many decades. However, neither the protective antigens of LVS nor the reason for its attenuation are known. This ignorance is hampering the development of both more rationally attenuated vaccine strains and acellular vaccines containing the protective antigens of the pathogen. All of the immunological data amassed to date indicates that cell mediated immunity directed against pathogen specific peptides displayed by MHC molecules of infected host cells underlies the success of LVS. In other words the protective antigens of F. tularensis are proteins. Moreover, they appear to be specific to type A and B strains of the pathogen, since the closely related subspecies, F. novicida, like LVS infects mice, but doesn't render them immune to challenge with virulent isolates of the former two strains. Similarly, the genetic defects that render LVS attenuated likely translate into a failure to express certain critical proteinaceous virulence factors. Therefore, comparing the proteomes of virulent and attenuated strains of the pathogen, and of LVS with F. novicida could reveal both the basis for the attenuation of LVS and the identity of its protective antigens. These are the aims of the current application. Any putative protective antigens identified will be incorporated into our ongoing acellular vaccine development program partially funded through grant AI48474. The genes for any putative virulence factors identified herein will form the basis for a new R01 grant application. It will focus on mutating such genes in virulent strains of the pathogen, and testing the ensuing mutants for their ability to act as alternative live vaccines to LVS.